Characterization of cell surface changes occurring upon transformation and understanding of the biochemical mechanisms under-lying these alterations are the twin aims of the research project. Investigation has focused on membranes of chick embryo fibroblasts (CEFs) transformed by Rous sarcoma virus (RSV). Reversible cross-linking agents, e.g. o-phenanthroline/CuSO4 and dimethyl dithiobisbutyrimidate are being used to examine protein-protein interactions in membranes of normal CEFs. Analysis of cross-linked components is being performed on 2-dimensional SDS polyacrylamide gels. Several cross-linked bands have been observed, including the dimer and a multimer of the 250,000 MW major surface protein Z. Further studies are directed at extending these results, with particular reference to cross-linking of lectin receptors and cytoskeletal elements. The pattern of cross-linking of normal membranes will be compared with those from transformed cells in order to see how such protein-protein interactions may be altered by transformation. The major surface protein lost from CEFs upon transformation is Z. This protein is highly sensitive to proteases such as plasmin, which is generated in the culture medium from RSV-transformed CEFs. We find that such conditioned medium was able to cause partial loss of Z from normal cells. Addition of soy bean trypsin inhibitor prevented this loss. Some batches of conditioned medium were inactive in cleaving Z. Inactive batches were deficient in proteolytic activity as measured by other assays. The tumor promotor PMA has been reported to stimulate production of plasminogen activator in CEFs. Its effects on Z, morphology, and growth control have therefore been examined. PMA caused an 80-85% decrease in the amount of Z per cell, with loss occurring over the course of 3 days. After removal of PMA, Z returned to its original level. Restoration of Z, like loss, required 3 days. Other effects of PMA on CEFs included stimulation of cell number and 3H-deoxyglucose uptake in resting cells, 2-fold decrease in cell volume, and change to a more spindle-shaped and less flattened morphology.